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Team:Wisconsin-Madison
From 2013.igem.org
Expression, Purification, and Quality Control of Gibson Enzymes.
Gibson assembly is a DNA cloning technique that was pioneered in 2009 by Daniel Gibson (insert citation). This technique utilizes three enzymes: (1) a thermostable DNA ligase; (2) a 5’-exonuclease; and, (3) a thermostable DNA polymerase, in an isothermal reaction to assemble together DNA fragments containing 20-40 base pair overlaps. Gibson assembly is significantly simpler to perform than prior conventional methods, as it is carried out in a single step and affords greater flexibility than methods relying on the use of restriction enzymes. Because of its simplicity and versatility, it has quickly become a common technique in molecular biology across the world. However, the financial cost of the necessary enzymes can be quite expensive, potentially limiting the access of smaller research labs and primarily undergraduate institutions to this groundbreaking technique.
This summer we have gene synthesized, expressed, purified, and assayed the three enzymes involved in Gibson assembly. We have carefully documented our protocol for purifying these enzymes and testing their functional efficacy relative to commercial versions. Lastly, we have combined our purifed enzymes in appropriate ratios to perform Gibson assembly. We are in the process of carefully comparing
In addition to this, we have developed a protocol for making a gibson master mix using lab purified enzymes, as well as an assay using a red fluorescent protein to test the functionality of a gibson reaction mix.
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